Malfunction of the cytoplasmatic enzyme Cu/Zn superoxide dismutase 1 (SOD1) has been linked to several neurological disorders such as Parkinson's disease, Alzheimer's disease, familial amyotrophic lateral sclerosis (FALS). While much is known about the structure of SOD1 and its associated copper chaperone CCS (copper chaperone for superoxide dismutase), the mechanisms by which CCS, and ultimately SOD1, obtains its copper cargo are largely unknown. Enabled by the recent success of our laboratory in determining the structure of the human copper transporter hCTR1, we hypothesize that CCS can obtain copper through a direct interaction that involves both the transporter and, unexpectedly, the membrane. Based on a hypothetical model for a CCS:hCTR1:membrane ternary complex, the objective of the proposed work is to visualize these molecular interactions and to determine the role of bilayers in cellular copper distribution to CCS. Two specific Aims will be pursued in this work: exploiting the unique ability of electron microscopy to directly visualize membrane proteins in their native environment, Aim 1 is focused on obtaining an electron microscopic reconstruction of a complex of membrane-embedded hCTR1 with CCS. Aim 2 is focused on determining how CCS interacts with bilayers, and how these interactions are modulated by the copper-loading status of the chaperone. These studies will use both in vitro membrane-binding experiments of CCS and select mutants as well as in vivo experiments to test the physiological significance of membrane-interactions in CCS function. Towards accomplishing the set goals, two-dimensional crystals of a hCTR1:CCS complex have already been obtained, and preliminary reconstructions from negatively stained samples are consistent with the proposed model. Moreover, in vitro experiments using purified CCS and select mutants demonstrate that it binds to bilayers through electrostatic interactions and provide an initial map of the putative membrane-binding site. The work proposed in this application will allow completion of these analyses and is expected to firmly establish a new paradigm for the mechanism of intracellular copper distribution to CCS. The work also will yield the first direct structural data on any complex between an intact copper transporter and a chaperone, which will make a critical contribution to understanding the structural basis of cellular copper homeostasis.